Alphaviruses and flaviviruses are the causative agents of severe human and animal illnesses such as encephalitis, polyarthritis, and dengue fever, with millions of cases in humans per year. These viruses also include potential bioterrorist agents such as the alphavirus Venezuelan Equine Encephalitis virus (a class B agent). Both alphaviruses and flaviviruses infect cells via a low pH-dependent membrane fusion reaction. Striking similarities exist between the native structures of the alphavirus and flavivirus membrane fusion proteins. Fusion by these "class II" fusion proteins appears mechanistically quite different from that of the class I fusion proteins exemplified by influenza virus, in which the fusion protein forms a "trimer of hairpins" with a central coiled-coil. The class II fusion proteins convert from a native dimer to a highly stable, non-coiled-coil based homotrimer. The goal of this application is to determine the molecular mechanism of membrane fusion of the alphavirus Semliki Forest virus (SFV), a member of the class II viruses and a highly developed system to study membrane fusion. Four key questions in the fusion mechanism of the class II proteins will be addressed: 1. Do class II fusion proteins refold to a "hairpin" conformation in which the fusion peptide and transmembrane domains are at the same end of the molecule? 2. Does the SFV E1 fusion protein mediate fusion by the transition to the stable homotrimer or by posttrimer events? Class I fusion proteins harness the energy of the transition from their metastable native conformation to the stable final hairpin to carry out membrane fusion. Kinetic and inhibitor studies will be used to determine the mechanism by which the class II homotrimer mediates fusion. 3. What is the structure and mechanism of the E1 fusion peptide in the membrane? We will define the region of E1 that becomes membrane-bound at low pH and whether it inserts into the target or virus membranes. 4. What is the structural basis of the SFV E1 homotrimer? We will compare the properties of native E1 with the E1 homotrimer by biochemical, morphological, and structural analysis. Ultimately, molecular information on the class II fusion proteins will enable the design of specific antiviral therapies, and advance our general understanding of cellular and viral membrane fusion reactions.